The dynamics of intracellular pH are believed to be crucial for understanding the regulation mechanism of many physiological functions.1 Of the methods available to determine pH, optical methods have several advantages. These include a rapid response time and a high signal-to-noise ratio. Additionally, they are non-invasive, and they generally have excellent pH sensitivity. Since the first use of a trapped intracellular pH probe, 6-carboxyfluorescein, was described by Thomas et al.12,31 a large number of intracellular pH indicators has been reported.1, 4-11 However, these one-photon excited fluorescent pH indicators have serious limitations, e.g. the interference of autotluorescence and scattering from biological fluids and tissue, photodamage of the samples and photobleaching of the indicators, difficulty in analyzing intercellular or intracellular pH differences, and others. Recently, using two-photon fluorescence (2 PF) to measure the pH has gained attention.12-15 Advantages of using the two-photon approach include less scattering and deeper penetration in biological samples by using NIR excitation light, less photodamage and photobleaching, as well as the unique properties of obtaining 3D resolution.
In addition to normal fluorescence methods, a 2 PF indicator has also been employed to detect the pH at molecular level by using fluorescence correlation spectroscopy.14 In order to quantitatively measure pH, the pK, of the indicator needs to match with the pH of the experimental system. Since the pH in the cell cytosol is typically between 6.8 and 7.4, there is tremendous interest in the development of an efficient two-photon absorbing (2 PA), near-neutral, fluorescent pH indicator. However, near-neutral 2 PF pH indicators are rare and the 2 PA cross-sections of most commercial pH indicators in the NIR region are low.12-15 Only recently, one example of a pH indicator designed with emphasis on improving the two-photon absorptivity was reported by Charier et al.13 A relatively high value of 60 GM (1 GM=10−50 cm4 s photon−1) was described, but its pKa of 5.7 is too low for near-neutral biological applications.